The primary axis of the zebrafish embryo is established by transcriptional control exerted by the Wnt pathway effector beta-catenin, which activates expression of the homeobox gene encoding the transcriptional repressor Bozozok/Dharma/Nieuwkoid (hereafter called Boz). Boz functions by repressing ventral genes such as vent, vox, ved and bmp. We found that the E3 ubiquitin ligase Lnx-2b (previously known as Lnx-like) can bind and ubiquitylate Boz, targeting it for proteasomal degradation. The presence of Lnx-2b balances the organizer and ventral domains in the embryo, assuring appropriate axis formation and normal development. These studies point to the modulation of protein stability as a fundamental mechanism in the regulation of early embryogenesis in the zebrafish. We have further explored the mechanism by which Lnx-2b restricts the size of the organizer domain. We found that modulation of the stability of Boz by Lnx-2b, in conjunction with the nodal signaling factor Nrd1, also known as Squint, acts to define the expression domain of the organizer gene goosecoid at the gastrula stage. Knock-down of Lnx-2b or overexpression of Ndr1 expands gsc expression throughout the mesoderm, but not into the presumptive ectoderm in the animal region of the embryo. However, the concomitant reduction of lnx-2b expression and increase in Ndr1 expression cooperate to induce the expansion of the gsc domain throughout the entire mesodermal and ectodermal domains of the gastrula embryo. Thus in the normal embryo the wide expression of Lnx-2b together with the restricted expression of Ndr1 act together to limit the domain of gsc induction and organizer formation to a specific region at the dorsal side of the embryo. Formation of the caudal domain and tissue differentiation in this region, such as hematopoiesis, requires Wnt signaling, which leads to the expression of transcription factors in the Cdx family. Several Cdx factors occur in vertebrates;in zebrafish, Cdx4 is the key factor responsible for caudal tissue formation and for hematopoiesis. We found that the transcription factor E4f1 enhances the expression of Cdx4. The mechanism underlying this effect is the interaction of E4f1 with the Wnt effector molecule Tcf3. In the absence of Wnt signaling, Tcf3 is associated with co-repressors such a Groucho/TLE and HDAC, forming a repressor complex that keeps the cdx4 gene silent. E4f1 can de-repress the cdx4 gene by dissociating co-repressors from Tcf3, without interfering with the binding of Tcf3 to its cognate sites in the DNA. Lnx-2b counteracts this effect by stabilizing the co-repressor complex. These studies have revealed a novel mechanism that modulates Wnt signaling during embryogenesis, and is likely to contribute to a robust read-out of the Wnt gradient that determines formation of the caudal body axis. A group of non-involuting cells at the dorsal, or organizer, side of the zebrafish embryo gives rise to a structure called Kupffers vesicle that appears to be homologous to the node of mouse and chicken embryos. This structure is essential for the establishment of left/right asymmetry, apparently by supporting cilia-driven fluid flow within the vesicle that is associated with symmetry breakage. We have found that two genes, previously known for other functions n the embryo, have a role in the differentiation of the precursors of Kupffers vesicle, named dorsal forerunner cells (DFC). The transcription factor Sox17 is best studied for its expression in the endoderm and its role in endoderm formation, while the signaling factor Chordin (Chd) is a key player in mediating organizer function in axis formation. Sox17 was known to be expressed in the DFC, and we showed that Chd as well is expressed in these cells. Using a targeted method for delivering antisense morpholino oligonucleotides to the embryo we showed that both Sox17 and Chd are critically required for the differentiation of DFC to the lining of Kupffers vesicle, and consequently for the establishment of left/right asymmetry in the embryo. A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as the pharyngeal arches. We found that the BTB-domain containing protein Kctd15 that is first expressed in the embryo in the neural plate border, is an important factor in regulating the domain in which neural crest forms. Overexpression of Kctd15 strongly inhibits neural crest specification, while attenuation of Kctd15 expression leads to expansion of the neural crest precursor domain. We have shown that anterior placodal domains are expanded after Kctd15 overexpression, supporting the idea that Kctd15 limits the extent of the neural crest domain and prevents incursion of the placodal domain by the neural crest. Among genes identified as differentially expressed in the pineal gland we noted a gene encoding a homolog of the Unc119 protein family. Whereas two Unc119 homologs are known in humans, the gene we isolated is the third family member noted in zebrafish;accordingly we named this protein Unc119c. The unc119c gene is specifically expressed in the pineal, with a low level of expression in the retina. In fish the pineal is a photosensitive organ and shows many similarities in gene expression to the retina. Unc119 proteins interact with small GTPases of the Arl3 family, and we have shown that Unc119c binds to Arl3l2 when both are coexpressed in heterologous cells. The biological function of Unc119c is being investigated by morpholino antisense oligonucleotide injection to inhibit its expression in the zebrafish embryo. Med12 is a component of the Mediator, a large complex involved in the transcriptional activation of many genes. With some variations, the Mediator complex and its components are conserved from yeast to humans. We have previously characterized a mutation, named kohtalo, that affects Med 12. Multiple tissues in the embryo develop abnormally in kohtalo embryos. Recently we have studied the nature of hindbrain patterning in this mutant. The hindbrain is segmented into units named rhombomeres, which are characterized by the expression of specific marker genes. Rhombomeres also have substructure that can be visualized by marker gene expression. We found that segmentation takes place in kohtalo mutants, but rhombomere boundary cells do not form. This finding confirms other results suggesting that segmentation per se does not require boundary cell differentiation, and further illustrates the specific effects that may arise in development as a result of loss of a widely expressed transcriptional regulatory molecule.